Many genetic diseases are difficult or impossible to treat with small molecule drugs or with enzyme replacement therapy as with Gaucher's disease. Recent advances in biology have raised the possibility that gene therapy, replacement of a defective gene with a normal gene, may be possible. Thus researchers have been studying several promising methods to deliver normal copies of genes to cells containing defective ones.
One promising method of gene delivery to humans is to use a plasmid DNA/lipid complex. The plasmid, a closed circular form of bacterial DNA, contains the genetic material needed to correct a genetic defect.
In some cases, large quantities of plasmid DNA ("pDNA") will be required to provide effective treatment to a population of patients afflicted with certain types of genetic diseases. Thus, recovery from the process must be high and the process must be scaleable to allow for efficient, cost effective production. Furthermore, the plasmid DNA will have to be very highly purified to allow for repeat dose administration of the complex to these patients. Because the plasmids used in clinical applications are produced typically by bacteria, e.g., E. Coli, the purification process must efficiently separate bacterial endotoxin, protein, and genomic (chromosomal) DNA from plasmid DNA, at very large scale. Because bacterial chromosomal DNA and plasmid DNA are so similar the process needs to be particularly effective at removing this contaminant.
Current methods described in the literature, such as cesium chloride centrifugation, chromatography on hydroxyapatite, or chromatographic methods based on reverse phase or anion exchange HPLC have limitations that would make purification of kilogram quantities difficult, if not impossible.
Purification of pDNA by centrifugation using cesium chloride involves use of toxic and carciongenic compounds, making this method unsuitable for purification of products for use in humans. Further, this technique could not be scaled to kilogram quantities per run.
Hydroxyapatite chromatography suffers from drawbacks as well. Lot variability of hydroxyapatite media would make consistent purification of plasmid DNA difficult. Further, because hydroxyapatite has a double positive charge and cannot be deprotonated, the resin cannot be cleaned, making it unusuable as a technique for purifying clinical grade material.
HPLC (high pressure liquid chromatography) techniques also suffer from scaleability problems. HPLC columns capable of purifying kilogram quantities of pDNA are not currently manufactured.
Thus, new and better methods continue to be sought for purifying pDNA and which are scaleable to produce kilogram quantities of plasmid DNA of sufficient purity to be used as a therapeutic in humans.